1. Field of the Invention
The present invention relates to a method for modifying a chromosome of yeast, employing yeast chromosome splitting vectors. More specifically, the present invention relates to a method for modifying a chromosome, employing two linear chromosome splitting vectors.
2. Description of the Related Art
The development of recombinant DNA technology has led to the development of techniques for modifying or manipulating genes as desired, and has made it possible to introduce modified genes into various organisms.
Techniques for modifying DNA include site-specific mutagenesis, treatments with restriction enzymes, and PCR. However, these techniques are only applicable to genes having a limited size such as about 10 kbp, at most about 40 kbp. Development of techniques for modifying large size DNA such as a chromosome has just begun.
Yeast is a useful organism that has been used in the fermentation industry for a long time. The chromosome of yeast can be modified by chromosome modification techniques such as splitting or loss of a chromosome so as to obtain information about, for example, the necessary number, length, and an amount of chromosomes. Based on such information, principle of yeast genome function and organization is expected to be constructed. If this principle is constructed, it may be possible that a desired substance can be produced efficiently by yeast in which unnecessary genes, for example, energy consuming genes are removed. Furthermore, if a desired region of a chromosome can be transferred into another yeast, the chromosome function of yeast can be analyzed so as to breed useful yeast.
DNA libraries including a large size DNA (e.g., 200 kbp or more) of plant chromosomes have been prepared by using, for example, a yeast artificial chromosome vector (YAC vector). If this large size DNA can be separated or manipulated as desired in accordance with various purposes, it may be possible to accelerate a development of plant biotechnology including breeding of useful transgenic plants that have not existed before. Further, it may also be possible to accelerate a development of basic biology for animals and plants, for example, elucidation of structure and function of animal and plant chromosomes, speed-up of positional cloning, simple and easy assay for animal and plant gene function, and construction of artificial chromosomes for animals or plants.
In this context, it has been attempted to manipulate chromosomes, for example, to split or loss of chromosomes of yeast or animal or plant. In order to stabilize a chromosome in yeast, it is necessary that a telomere sequence is present at a terminal of the chromosome. For this reason, the conventional technique for splitting a chromosome in yeast utilizes a phenomenon that the telomere sequence of the yeast is added to the telomere sequence derived from Tetrahymena rDNA (hereinafter referred to as “Tr sequence”) at a high frequency in order to stabilize the split chromosome (see Shampay et al. (1984) Nature (London) 310, 154-157). FIGS. 15A to 15D show the outline of this conventional technique for splitting a chromosome. This technique is based on the following phenomenon: when a vector haboring centromere (C), telomere repetitive sequence (Tr-Tr) derived from Tetrahymena rDNA that are placed in opposite directions to each other, a selection marker (M) for a yeast transformation, and any target sequence is introduced into a yeast chromosome by transformation (FIG. 15A), homologous recombination occurs within the target sequence (FIG. 15B). Then, telomere resolution occurs at the Tr-Tr sequence that has been introduced into the chromosome so as to split the chromosome (FIG. 15C), followed by addition of telomere sequence of the yeast to the dissociated terminal (Tr sequence) of the split chromosomes (FIG. 15D).
FIGS. 16A and 16B show the Tr sequence derived from Tetrahymena. The Tr sequence consists of about 700 bp, in which an AT rich portion of about 400 bp is followed by a repetitive sequence element of 5′-CCCCAA-3′ (C4A2) of about 300 bp. It seems that this repetitive sequence serves as a signal to which the telomere sequence of the yeast can be added effectively (FIG. 16A). It has been reported that the telomere sequence of the yeast can be added to a terminal sequence (C4A2)6 consisting of only 6 repeats of C4A2 (FIG. 16B) (see Murray et al., (1988) Mol. Cell. Biol. 8(11), 4642-4650).
There has been a report of an attempt to split a yeast chromosome utilizing the phenomenon shown in FIG. 16A, for example, using a yeast chromosome vector pCSV1 having a centromere gene (CEN4) of the yeast, a marker gene (URA3), two telomere sequences that are placed in opposite directions each other and a HIS3 gene as stuffer DNA between the two telomere sequences (see Japanese Laid-Open Patent Publication No. 10-84945).
FIG. 17 shows the splitting of a yeast chromosome with this splitting vector pCSV1. In FIG. 17, the pCSV1 has CEN4, URA3 (not shown), Tr sequences that are placed in opposite directions each other, and a stuffer DNA (X) between the two Tr sequences. The splitting includes the steps of: (Step 1) introducing a target sequence (Y) into the splitting vector pCSV1, wherein the target sequence (Y) has a splitting site; (Step 2) removing the stuffer DNA (X); (Step 3) cyclizing the plasmid from which the stuffer DNA (X) has been removed; and (Step 4) cleaving the cyclized plasmid at the target sequence (Y) to obtain a linear splitting vector so as to introduce it into a homologous region of a yeast chromosome. It seems that by introducing the linear splitting vector into a yeast cell, homologous recombination occurs within the target sequence, and then the oppositely-oriented telomere sequences are resolved so as to split the chromosome.
However, in the above-described method, a complicated four step procedure is necessary, and in this method, the following time-consuming work is required: (1) it is necessary to clone the target sequence to the splitting vector for every splitting; (2) the vector used in Step 1 to which the target sequence Y is introduced and the vector used in Step 3 that is re-cyclized have to be introduced into bacterial cells for amplification; and (3) the target sequence is cleaved at only one site with a restriction enzyme in order to increase an efficiency of oriented integration at a homologous region. Thus, in this method, a plurality of complicated steps is required and it takes much time for the splitting of chromosome.
Therefore, it was attempted to split a chromosome by preparing a linear splitting vector (A) having a telomere sequence (i)—a centromere sequence of a yeast chromosome—a target sequence (i) in this order, and a linear splitting vector (B) having a target sequence (b)—a marker gene sequence—a telomere sequence (ii) in this order, and introducing them into a yeast. When these two linear splitting vectors were introduced into the yeast, splitting of yeast chromosome was confirmed. These two splitting vectors was obtained from a vector having a target sequence (δ)—CEN—a Tr sequence and a vector having a target sequence (δ)—a marker gene—a Tr sequence, as shown in FIG. 18A. Therefore, there is no need for complicated steps that are required in the conventional method using the chromosome splitting vectors, for example, as shown in FIG. 17.
However, the two splitting vectors could not be amplified by PCR (see FIG. 18C). PCR was performed in order to obtain linear splitting vectors (A) and (B) using a plasmid that contain Tr sequence as a template as shown in FIG. 18B. The PCR products 7 and 8 were analyzed and it was found that non-specific amplification had occurred as shown in FIG. 18C. One of the reasons why the two splitting vectors could not specifically be amplified by PCR is that there is a repetitive sequence of about 300 bp in the Tr sequence, as described above. For this reason, even with the method using the two splitting vectors, which is much simpler than the method shown in FIG. 17, an operation of amplifying and cleaving a cyclic vector and collecting DNA fragments still has to be performed to obtain the splitting vectors, which is still complicated.
Therefore, it is desirable to develop a simpler technique for manipulating, or isolation of the large size DNA such as, in particular, chromosomes, using a yeast as a host.